The majority of biotechnology processes for producing pharmaceutical or diagnostic products involve the purification of proteins and peptides from a variety of sources. Those include bacteria, yeast and mammalian cell culture fluids, or extracts from naturally occurring tissue (Expanded Bed Adsorption: Principles and Methods, Pharmacia Biotech, ISBN 91-630-5519-8).
The initial purification of a protein or peptide is often via the use of adsorption chromatography on a conventional packed bed of solid support adsorbent. This frequently requires clarification of the crude cell culture or tissue mixture before application onto the chromatography column (Pharmacia Biotech, supra).
Standard techniques used for removal of cells and/or cell debris include centrifugation and microfiltration, which may be used separately or in combination. Packed bed chromatography is problematic in that clogging of the bed occurs readily when passing crude material over the bed, making prechromatographic centrifugation and/or microfiltration necessary and adding to the time and cost of product recovery. Batch adsorption chromatography is a one-step adsorption process of the protein product to a resin in a stirred tank. However, batch adsorption requires large amounts of resin, thereby greatly increasing the cost of recovery (Pharmacia Biotech, supra).
Expanded bed adsorption (EBA) chromatography is useful for the initial recovery of target proteins from crude feed-stock or cell culture. The process steps of clarification, concentration and initial purification can be combined into one unit operation, providing increased process economy due to a decreased number of process steps, increased yield, shorter overall process time, reduced labor cost, and reduced materials cost. In EBA chromatography an adsorbent is expanded and equilibrated by applying an upward liquid flow to the column (see FIG. 1). A stable fluidized bed is formed when the adsorbent particles are suspended in equilibrium due to the balance between particle sedimentation velocity and upward liquid flow velocity. During this phase column adapter is positioned in the upper part of the column and a crude cell mixture is applied to the expanded bed with an upward flow. Target proteins in the mixture are bound to the adsorbent while cell debris and other contaminants pass through unhindered. Weakly bound material is washed from the expanded bed using upward flow of a wash buffer. Cell debris and suspended solids in the column may be flushed to prevent contamination of the elution pool by particulate material (Draeger, N. M. and Chase, H. A., Bioseparation 2:67-80 (1991); Chang, Y. K. et al., Biotechnology and Bioengineering, 48:355-366 (1995); and Chang, Y. K. and Chase, H. A., Biotechnology and Bioengineering, 49:204-216 (1996)).
Following a wash step, flow through the column is stopped and the adsorbent is allowed to settle in the column. The column adapter is then lowered to the surface of the sedimented bed. Flow is reversed and the captured proteins are eluted from the sedimented bed using an appropriate buffer. The eluate contains the target protein in a reduced elution pool volume, partially purified in preparation for packed bed chromatography (Pharmacia Biotech, supra). Flow reversal during protein purification was used to minimize sample band diffusion after loading a dense sample solution (such as an ammonium sulfate protein precipitate) onto a column, but inconveniently required actual inversion of the column to change the direction of flow through the column (Scopes, Robert K., Ch. 8, "Separation in Solution," in Protein Purification: Principles and Practice, Springer-Verlag, NY (1994), pp. 242-249).
There is a need for a simple, cost-effective method to reduce elution pool volume, increase purity of a sample molecule collected from a chromatographic method and increase the concentration of the sample molecule as it is collected from a chromatographic method without, for example, waiting for the adsorbent bed to settle, without a need for a moving column adapter, and without manual column inversion. Such a method would save equipment costs and time for sample purification while improving the level of purity of the target protein or peptide.